A short-pulse saturation-recovery electron spin resonance technique has been used to study the effect of polar carotenoid-lutein and cholesterol on interactions of 14N:15N stearic acid spin-label pairs in fluid-phase phosphatidylcholine (PC) membranes. Bimolecular collisions for pairs consisting of various combinations of [14N]-16-, [14N]-10-, [14N]-7-, or [14N]-5-doxylstearate and [15N]-16-doxylstearate in dimyristoyl-PC (DMPC) or egg yolk PC (EYPC) membranes were measured at 27x C. In the absence and presence of lutein or cholesterol for both lipid systems, the collision rates were ordered as 16:5<16:7<16:10<16:16. For all spin-label pairs studied, interaction frequencies were greater in DMPC than in EYPC. Polar carotenoid-lutein reduces the collision frequency for all spin-label pairs, while cholesterol reduces the collision frequency for 16:5 and 16:7 pairs and increases the collision frequency in the membrane center for 16:10 and 16:16 pairs. The presence of unsaturated alkyl chains greatly reduces the effect of lutein, but magnifies the effect of cholesterol in the membrane center. The observed differences in the effects of these modifiers on alkyl-chain bending results from differences in the structure of cholesterol and polar carotenoid and from different localizations within the lipid bilayer membrane. These studies further confirm the occurrence of vertical fluctuations of alkyl chain ends toward the bilayer surface. The decrease of the frequency of alkyl chains vertical fluctuation may be an addition mechanism of carotenoid protective activity against the lipid peroxidation, which should be effective in plant and animal cells in the light as well as in the dark.